PNA-DNA-PNA chimeric macromolecules

ABSTRACT

Macromolecules are provided that have increased nuclease resistance, increasing binding affinity to a complementary strand, and that activate RNase H enzyme. The macromolecules have the structure PNA-DNA-PNA where the DNA portion is composed of subunits of 2′-deoxy-erythro-pento-furanosyl nucleotides and the PNA portions are composed of subunits of peptide nucleic acids. Such macromolecules are useful for diagnostics and other research purposes, for modulating protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to therapeutics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 08/877,317, filedon Jun. 17, 1997, which is division of application Ser. No. 08/158,352,filed on Nov. 24, 1993 (now U.S. Pat. No. 5,700,922), which is acontinuation-in-part of application serial number PCT/US92/11339, filedDec. 23, 1992 (now U.S. Pat. No. 5,623,065, issued Apr. 22, 1997),which, in turn, is a continuation-in-part of my prior application Ser.No. 07/814,961, filed Dec. 24, 1991 (now abandoned). Both of theforegoing patent applications are commonly assigned with thisapplication. This application is further related to application Ser. No.088,658, filed Jul. 2, 1993, entitled “Higher Order Structure andBinding of Peptide Nucleic Acids,” commonly assigned in part with thisapplication. That application is a continuation-in-part of applicationSer. No. 054,363, filed Apr. 26, 1993, entitled “Novel Peptide NucleicAcids,” which in turn is a continuation-in-part of application PCTEP/91/01219, filed May 19, 1992. The disclosures of each of theseapplications are herein incorporated by reference.

FIELD OF THE INVENTION

This invention is directed to the synthesis and use of chimericmolecules having a PNA-DNA-PNA structure wherein each “PNA” is a peptidenucleic acid and the “DNA” is a phosphodiester, phosphorothioate orphosphorodithioate 2′-deoxyoligonucleotide. In a cell, a cellularextract or a RNase H containing diagnostic test system, a chimericmacromolecule of the invention having a base sequence that ishybridizable to a RNA target molecule can bind to that target RNAmolecule and elicit a RNase H strand cleavage of that RNA targetmolecule.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals includingmost disease states, are effected by proteins. Such proteins, eitheracting directly or through their enzymatic functions, contribute inmajor proportion to many diseases in animals and man. Classicaltherapeutics has generally focused upon interactions with such proteinsin an effort to moderate their disease causing or disease potentiatingfunctions. Recently, however, attempts have been made to moderate theactual production of such proteins by interactions with messenger RNA(mRNA) or other intracellular RNA's that direct protein synthesis. It isgenerally the object of such therapeutic approaches to interfere with orotherwise modulate gene expression leading to undesired proteinformation.

Antisense methodology is the complementary hybridization of relativelyshort oligonucleotides to single-stranded RNA or single-stranded DNAsuch that the normal, essential functions of these intracellular nucleicacids are disrupted. Hybridization is the sequence specific hydrogenbonding via Watson-Crick base pairs of the heterocyclic bases ofoligonucleotides to RNA or DNA. Such base pairs are said to becomplementary to one another.

Naturally occurring events that provide for the disruption of thenucleic acid function, as discussed by Cohen in Oligonucleotides:Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton,Fla. (1989) are thought to be of at least two types. The first ishybridization arrest. This denotes the terminating event in which anoligonucleotide inhibitor binds to a target nucleic acid and thusprevents, by simple steric hindrance, the binding of essential proteins,most often ribosomes, to the nucleic acid. Methyl phosphonateoligonucleotides (see, e.g., Miller, et al., Anti-Cancer Drug Design1987, 2, 117) and α-anomer oligonucleotides are the two most extensivelystudied antisense agents that are thought to disrupt nucleic acidfunction by hybridization arrest.

In determining the extent of hybridization arrest of an oligonucleotide,the relative ability of an oligonucleotide to bind to complementarynucleic acids may be compared by determining the melting temperature ofa particular hybridization complex. The melting temperature (T_(m)), acharacteristic physical property of double helixes, denotes thetemperature in degrees centigrade at which 50% helical (hybridized)versus coil (unhybridized) forms are present. T_(m) is measured by usingthe UV spectrum to determine the formation and breakdown (melting) ofhybridization. Base stacking which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the binding of thestrands. Non-Watson-Crick base pairing, i.e. base mismatch, has a strongdestabilizing effect on the T_(m).

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of the targeted RNA by intracellularRNase H. The mechanism of such RNase H cleavages requires that a2′-deoxyribofuranosyl oligonucleotide hybridize to a targeted RNA. Theresulting DNA-RNA duplex activates the RNase H enzyme; the activatedenzyme cleaves the RNA strand. Cleavage of the RNA strand destroys thenormal function of the RNA. Phosphorothioate oligonucleotides are oneprominent example of antisense agents that operate by this type ofterminating event. For a DNA oligonucleotide to be useful for activationof RNase H, the oligonucleotide must be reasonably stable to nucleasesin order to survive in a cell for a time sufficient for the RNase Hactivation.

Several recent publications of Walder, et al. further describe theinteraction of RNase H and oligonucleotides. Of particular interest are:(1) Dagle, et al., Nucleic Acids Research 1990, 18, 4751; (2) Dagle, etal., Antisense Research And Development 1991, 1, 11; (3) Eder, et al.,J. Biol. Chem. 1991, 266, 6472; and (4) Dagle, et al., Nucleic AcidsResearch 1991, 19, 1805. In these papers, Walder, et al. note that DNAoligonucleotides having both unmodified phosphodiester internucleosidelinkages and modified, phosphorothioate internucleoside linkages aresubstrates for cellular RNase H. Since they are substrates, theyactivate the cleavage of target RNA by the RNase H. However, the authorsfurther note that in Xenopus embryos, both phosphodiester linkages andphosphorothioate linkages are also subject to exonuclease degradation.Such nuclease degradation is detrimental since it rapidly depletes theoligonucleotide available for RNase H activation. As described inreferences (1), (2), and (4), to stabilize their oligonucleotidesagainst nuclease degradation while still providing for RNase Hactivation, Walder, et al. constructed 2′-deoxy oligonucleotides havinga short section of phosphodiester linked nucleotides positioned betweensections of phosphoramidate, alkyl phosphonate or phosphotriesterlinkages. While the phosphoramidate containing oligonucleotides werestabilized against exonucleases, in reference (4) the authors noted thateach phosphoramidate linkage resulted in a loss of 1.6° C. in themeasured T_(m) value of the phosphoramidate containing oligonucleotides.Such decrease in the T_(m) value is indicative of an undesirabledecrease in the hybridization between the oligonucleotide and its targetstrand.

Other authors have commented on the effect such a loss of hybridizationbetween an antisense oligonucleotide and its targeted strand can have.Saison-Behmoaras, et al., EMBO Journal 1991, 10, 1111, observed thateven through an oligonucleotide could be a substrate for RNase H,cleavage efficiency by RNase H was low because of weak hybridization tothe mRNA. The authors also noted that the inclusion of an acridinesubstitution at the 3′ end of the oligonucleotide protected theoligonucleotide from exonucleases.

U.S. Pat. No. 5,149,797 to Pederson et. al., that issued on Sep. 22,1992 describes further oligonucleotides that operate by a RNase Hmechanism. The oligonucleotides as claimed in this patent consist of aninternal segment composed of phosphorothioate nucleotides flanked bymethyl phosphonate, phosphoromorpholidates, phosphoropiperazidates orphosphoramidates. Since all of the components of these oligonucleotides,i.e. phosphorothioate, methyl phosphonates, phosphoromorpholidates,phosphoropiperazidates or phosphoramidates when used as oligonucleotidelinkages individually decrease the hybridization between theoligonucleotide and its target strand, the comments of Saison-Behmoaraset al., could be equally applicable to the oligonucleotides described inthis patent.

While it has been recognized that cleavage of a target RNA strand usingan antisense oligonucleotide and RNase H would be useful, nucleaseresistance of the oligonucleotide and fidelity of the hybridization arealso of great importance. Heretofore, there have been no suggestion inthe art of methods or materials that could both activate RNase H whileconcurrently maintaining or improving hybridization properties andproviding nuclease resistance even though there has been a long feltneed for such methods and materials. Accordingly, there remains along-felt need for such methods and materials.

OBJECTS OF THE INVENTION

It is an object of this invention to provide chimeric macromoleculesthat hybridize with a target strand with improved binding affinity.

It is a further object to provide chimeric macromolecules that havestability against nuclease degradation.

A still further object is to provide chimeric macromolecules thatactivate RNase H for target strand cleavage.

A still further object is to provide research and diagnostic methods andmaterials for assaying cellular states in vitro and bodily states,especially diseased states, in animals.

Another object is to provide therapeutic and research methods andmaterials for the treatment of diseases through modulation of theactivity of a target RNA.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with this invention there are provided macromoleculesformed from peptide nucleic acids and 2′-deoxyoligonucleotides. Suchmacromolecules have the structure: PNA-DNA-PNA, wherein the PNA portionsare peptide nucleic acid sequences and the DNA portion is aphosphodiester, phosphorothioate or phosphorodithioate2′-deoxyoligonucleotide sequence. The PNA portions of the macromoleculeis believed to provide increased nuclease resistance and increasedbinding affinity of the macromolecule to target RNAs. The2′-deoxyoligonucleotide portion is believed to elicit a RNase H responseand cleavage of a RNA target strand.

The 2′-deoxyoligonucleotide, i.e. DNA, portions of the macromolecules ofthe invention are oligonucleotide segments formed from nucleotide unitsthat have 2′-deoxy-erythro-pento-furanosyl sugar moieties. Eachnucleotide includes a nucleobase attached to a2′-deoxy-erythro-pentofuranosyl sugar moiety of the nucleotide. Thenucleotides are linked together and/or to other moieties byphosphodiester linkages, phosphorothioate linkages and/orphosphorodithioate linkages. In certain preferred macromolecules of theinvention each of the nucleotides of the 2′-deoxyoligonucleotide portionof the macromolecule are linked together by phosphorothioate linkages.In other preferred embodiments, the nucleotides of the2′-deoxyoligonucleotide portion are linked together by phosphodiesterlinkages and in even further preferred embodiments, a mixture ofphosphodiester and phosphorothioate linkages link the nucleotide unitsof the 2′-deoxyoligonucleotide together.

The peptide nucleic acid portions of the macromolecules increase thebinding affinity of the macromolecule to a complementary strand ofnucleic acid. It further provides for nuclease stability of themacromolecule against degradation by cellular nucleases. Selecting the2′-deoxyoligonucleotide portion of the macromolecule to include one ormore or all phosphorothioate or phosphorodithioate linkages providesfurther nuclease stability to the macromolecules of the invention.

The PNA portions of the macromolecules of the invention are made up ofunits comprising a N-(2-aminoethyl)-glycine or analogues thereof havinga nucleobase attached thereto via a linker such as a carboxymethylmoiety or analogues thereof to the nitrogen atom of the glycine portionof the unit. The units are coupled together via amide bonds formedbetween the carboxyl group of the glycine moiety and the amine group ofthe aminoethyl moiety. The nucleobase can be one of the four commonnucleobases of nucleic acids or they can include other natural orsynthetic nucleobases.

In preferred macromolecules of the invention the PNA-DNA-PNA structureis formed by connecting together the respective N-(2-aminoethyl)glycinePNA units and the respective 2′-deoxy-erythro-pentofuranosyl sugarphosphate DNA units. Thus the nucleobases of the PNA portion of themacromolecules of the invention are carried on a backbone composed ofN-(2-aminoethyl)glycine PNA units and the nucleobases of the DNA portionof the macromolecules of the invention are carried on a backbonecomposed of 2′-deoxy-erythro-pentofuranosyl sugar phosphate units.Together the nucleobases of the PNA portions and the nucleobases of theDNA portion of the macromolecules of the invention are connected bytheir respective backbone units in a sequence that is hybridizable to acomplementary nucleic acid, as for instance, a targeted RNA stand.

In preferred macromolecules of the invention the PNA and the DNAportions are joined together with amide linkages. In such preferredmacromolecule of the invention the macromolecule is of the structure:

PNA-(amide link)-DNA- (amide link)-PNA.

Other linkages that can be used to join the PNA and the DNA portionsinclude amine linkages and ester linkages.

The macromolecules of the invention preferably comprise from about 9 toabout 30 total nucleobase bearing subunits. It is more preferred thatthe macromolecules comprise from about 15 to about 25 nucleobase bearingsubunits. In order to elicit a RNase H response, as specified above,within this total overall sequence length of the macromolecule will be asub-sequence of greater than 3 but preferably five or more consecutive2′-deoxy-erythro-pentofuranosyl containing nucleotide subunits.

Preferred nucleobases of the invention for both the peptide nucleic acidand the 2′-deoxynucleotide subunits include adenine, guanine, cytosine,uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl andother alkyl adenines, 2-propyl and other alkyl adenines, 5-halo uracil,5-halo cytosine, 5-propynyl uracil, 5-propynyl cytosine, 7-deazaadenine,7-deazaguanine, 7-deaza-7-methyl-adenine, 7-deaza-7-methyl-guanine,7-deaza-7-propynyl-adenine, 7-deaza-7-propynyl-guanine and other7-deaza-7-alkyl or 7-aryl purines, N2-alkyl-guanine,N2-alkyl-2-amino-adenine, purine 6-aza uracil, 6-aza cytosine and 6-azathymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo adenine,8-amino-adenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8 substituted adenines and 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8 substituted guanines, other aza and deaza uracils, other aza anddeaza thymidines, other aza and deaza cytosine, aza and deaza adenines,aza and deaza guanines or 5-trifluoromethyl uracil and5-trifluorocytosine.

The invention also provides methods of treating an organism having adisease characterized by the undesired production of a protein. Thesemethods include contacting the organism with a macromolecule having asequence of nucleobases capable of specifically hybridizing to acomplementary strand of nucleic acid. The methods further includinghaving a portion of the nucleobases comprise peptide nucleic acidsubunits (PNA units) and the remainder of the nucleobases comprise2′-deoxynucleotide subunits (DNA units). The peptide nucleic acidsubunits and the deoxynucleotide subunits are joined together to form amacromolecule of the structure PNA-DNA-PNA where the PNAs are peptidenucleic acids and DNA is an 2′-deoxyoligonucleotide.

Further in accordance with this invention there are providedcompositions including a pharmaceutically effective amount of amacromolecule having a sequence of nucleobases capable of specificallyhybridizing to a complementary strand of nucleic acid. The methodsfurther include having a portion of the nucleobases comprise peptidenucleic acid subunits (PNA units) and the remainder of the nucleobasescomprise 2′-deoxy-nucleotide subunits (DNA units). The peptide nucleicacid subunits (the PNAs) and the deoxynucleotides subunits (the DNA) arejoined together to form a macromolecule of the structure PNA-DNA-PNA.The composition further include a pharmaceutically acceptable diluent orcarrier.

Further in accordance with this invention there are provided methods forin vitro modification of a sequence specific nucleic acid includingcontacting a test solution containing an RNase H enzyme and said nucleicacid with a PNA-DNA-PNA macromolecule, as defined above.

There are also provided methods of concurrently enhancing hybridizationand RNase H enzyme activation in an organism that includes contactingthe organism with a PNA-DNA-PNA macromolecule, as defined above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chemical schematic illustrating solid phase synthesis ofcertain compounds of the invention (SEQ ID NO: 1);

FIG. 2 is a chemical schematic illustrating solid phase synthesis ofcertain compounds of the invention (SEQ ID NO: 2);

FIG. 3 is a chemical schematic illustrating solution phase synthesis ofcertain compounds of the invention;

FIG. 4 is a chemical schematic illustrating solution phase synthesis ofcertain compounds of the invention;

FIG. 5 is a chemical schematic illustrating solid phase synthesis ofcertain compounds of the invention; and

FIG. 6 is a chemical schematic illustrating solution phase synthesis ofcertain compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the objects of this invention, novel macromoleculesare provided that, at once, have increased nuclease resistance,increased binding affinity to complementary strands and that aresubstrates for RNase H. The macromolecules of the invention areassembled from a plurality of peptide nucleic acid subunits (PNAsubunits) and a plurality of 2′-deoxynucleotide subunits (DNA subunits).They are assembled into a macromolecule of the structure: PNA-DNA-PNA.Each peptide nucleic acid subunit and each 2′-deoxynucleotide subunitincludes a nucleobase that is capable of specifically hybridizing withlike nucleobases on a target RNA molecules or other target moleculesincluding DNA molecules and proteins.

The peptide nucleic acid portions of the macromolecules of the inventionbestow increased nuclease resistance to the macromolecules of theinvention. Further, these same peptide nucleic acid portions bestowincreased binding affinity of the macromolecules of the invention to acomplementary strand of nucleic acid. The 2′-deoxynucleotide portion ofthe macromolecules of the invention each include a2′-deoxy-erythro-pentofuranosyl group as their sugar moiety.

In conjunction with the above guidelines, each of the 2′-deoxynucleotidesubunits can be a “natural” or a “synthetic” moiety. Thus, in thecontext of this invention, the term “oligonucleotide” in a firstinstance refers to a polynucleotide formed from a plurality of joinednucleotide units. The nucleotides units are joined together via nativeinternucleoside, phosphodiester linkages. The nucleotide units areformed from naturally-occurring bases and2′-deoxy-erythro-pentofuranosyl sugars groups. The term“oligonucleotide” thus effectively includes naturally occurring speciesor synthetic species formed from naturally occurring nucleotide units.

The term oligonucleotide is intended to include naturally occurringstructures as well as non-naturally occurring or “modified”structures—including modified base moieties that function similarly tonatural bases. The nucleotides of the 2′-deoxyoligonucleotide portion ofthe macromolecule can be joined together with other selected syntheticlinkages in addition to the natural phosphodiester linkage. These otherlinkages include phosphorothioate and phosphorodithioate inter-sugarlinkages. Further suggested as suitable linkages are phosphoroselenateand phosphorodiselenate linkages. The base portion, i.e., the nucleobaseof the 2′-deoxynucleotides, can include the natural bases, i.e. adenine,guanine, cytosine, uracil or thymidine. Alternately they can includedeaza or aza purines and pyrimidines used in place of natural purine andpyrimidine bases; pyrimidine bases having substituent groups at the 5 or6 position; purine bases having altered or replacement substituentgroups at the 2, 6 or 8 positions. They may also comprise othermodifications consistent with the spirit of this invention. Such2′-deoxyoligonucleotides are best described as being functionallyinterchangeable with natural oligonucleotides (or synthesizedoligonucleotides along natural lines), but which have one or moredifferences from natural structure. All such 2′-deoxy-oligonucleotidesare comprehended by this invention so long as they function effectivelyin the macromolecule to elicit the RNase H cleavage of a target RNAstrand.

In one preferred embodiment of this invention, nuclease resistancebeyond that confirmed by the peptide nucleic acid portion of themacromolecule is achieved by utilizing phosphorothioate internucleosidelinkages. Contrary to the reports of Walder, et al. note above, I havefound that in systems such as fetal calf serum containing a variety of3′-exonucleases, modification of the internucleoside linkage from aphosphodiester linkage to a phosphorothioate linkage provides nucleaseresistance.

Brill, et al., J. Am. Chem. Soc. 1991, 113, 3972, recently reported thatphosphorodithioate oligonucleotides also exhibit nuclease resistance.These authors also reported that phosphorodithioate oligonucleotide bindwith complementary deoxyoligonucleotides, stimulate RNase H andstimulate the binding of lac repressor and cro repressor. In view ofthese properties, phosphorodithioates linkages also may be use in the2′-deoxyoligonucleotide portion of the macromolecules of the invention.The synthesis of phosphorodithioates is further described by Beaton, et.al., Chapter 5, Synthesis of oligonucleotide phosphorodithioates, page109, Oligonucleotides and Analogs, A Practical Approach, Eckstein, F.,Ed.; The Practical Approach Series, IRL Press, New York, 1991.

When increased nuclease resistance is conferred upon a macromolecule ofthe invention by the use of a phosphorothioate or phosphorodithioatesinternucleotide linkages, such linkages will reside in eachinternucleotide sites. In other embodiments, less than all of theinternucleotide linkages will be modified to phosphorothioate orphosphorodithioate linkages.

I have found that binding affinity of macromolecules of the invention isincreased by virtue of the peptide nucleic acid portions of themacromolecules. As for example the T_(m) of a 10 mer homothymidine PNAbinding to its complementary 10 mer homoadenosine DNA is 73° C. whereasthe T_(m) for the corresponding 10 mer homothymidine DNA to the samecomplementary 10 homoadenosine DNA is only 23° C.

Binding affinity also can be increased by the use of certain modifiedbases in both the nucleotide subunits that make up the2′-deoxyoligonucleotides of the invention and in the peptide nucleicacid subunits. Such modified bases may include 5-propynylpyrimidines,6-azapyrimidines, and N-2, N-6 and O-6 substituted purines including2-aminopropyladenine. Other modified pyrimidine and purine base are alsoexpected to increase the binding affinity of macromolecules to acomplementary strand of nucleic acid.

For use in preparing such structural units, suitable nucleobases includeadenine, guanine, cytosine, uracil, thymine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudo uracil), 4-thiouracil, 8-halo, amino, thiol, thiolalkyl,hydroxyl and other 8 substituted adenines and guanines,5-trifluoromethyl and other 5 substituted uracils and cytosines,7-methylguanine and other nucleobase such as those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, J. I. Kroschwitz, Ed. John Wiley &Sons, 1990 at pages 858-859 and those disclosed by Englisch, U. andGauss, D. H., Angewandte Chemie, International Edition 1991, 30, 613 areselected.

In order to elicit RNase H enzyme cleavage of a target RNA, amacromolecule of the invention must include a segment or sub-sequencetherein that is a DNA type segment. Stated otherwise, at least a portionof the macromolecules of the invention must be nucleotide subunitshaving 2′-deoxy-erythro-pentofuranosyl sugar moieties. I have found thata sub-sequence having more than three consecutive, linked2′-deoxy-erythro-pentofuranosyl-containing nucleotide subunits likely isnecessary in order to elicit RNase H activity upon hybridization of amacromolecule of the invention with a target RNA. It is presentlypreferred to have a sub-sequence of 5 or more consecutive2′-deoxy-erythro-pentofuranosyl containing nucleotide subunits in amacromolecule of the invention. Use of at least 7 consecutive2′-deoxy-erythro-pentofuranosyl-containing nucleotide subunits isparticularly preferred.

The overall length of the macromolecules of the invention can be from 3to hundreds of subunits long; however, since target specificity can beachieved with a much shorter molecule, a more practical maximum lengthwill be from about 30 to about 50 subunits long. An even more preferredmaximum length will be about 25 subunits long.

Depending upon the target, the minimum length of the macromolecule willvary from three to about fifteen total subunits. It has been found inpractice that in using antisense oligonucleotides generally a minimumlength of about 15 nucleotides is necessary to insure proper affinityupon hybridization. However as noted above, since the peptide nucleicacid subunits have a greater hybridization affinity for target moleculescompared to normal phosphodiester oligonucleotides that in turn have abetter affinity than phosphorothioate oligonucleotides, in using themacromolecules of the invention, a minimum length less than thatnormally use in practicing antisense binding with antisenseoligonucleotides can be expected. Taking these factors in toconsideration a preferred length of the macromolecules will be fromabout 3 to about 30 total subunits with a more preferred range fromabout 9 to about 25 subunits in length.

In the macromolecules of the invention, there will be one or moresequential DNA units interspaced between PNA units. To elicit a RNase Hresponse, as noted above preferably the DNA portion of the macromoleculewill have at least three 2′-deoxy-nucleotide units. In determining anupper range of the number of DNA units, consideration is given toseveral factors including overall length of the macromolecule, thephosphate linkage utilized, desired fidelity to a target sequence andother like factors. Normally, for economic considerations it isdesirable not to have more nucleobase units in the macromolecules of theinvention than is necessary to bind with specificity to a targetmolecule and, if desired, to elicit an RNase H response. For utilizationof the RNase H mechanism this number is generally about 5 nucleotides.Additionally since phosphorothioate and phosphorodithioate phosphatelinkage themselves exhibit nuclease resistance, with use of these twophosphate linkages, a longer stretch of DNA subunits can be utilizedcompared to phosphodiester subunits that must rely on the PNA portionsof the macromolecule for nuclease resistance. Taking these factors intoaccount, a particularly preferred working range includes macromoleculesof the invention is from 9 to about 28 subunits in length and havingfrom about five to about eight of those subunits being sequentiallocated 2′-deoxynucleotide subunits.

The mechanism of action of RNase H is recognition of a DNA-RNA duplexfollowed by cleavage of the RNA stand of this duplex. As noted in theBackground section above, others in the art have used modified DNAstrands to impart nuclease stability to the DNA strand. To do this theyhave used modified phosphate linkages that impart increased nucleasestability but concurrently detract from hybridization properties.

While I do not wish to be bound by theory in the macromolecules of theinvention, I have recognized that by positioning peptide nucleic acidunits at both ends of a 2′-deoxyoligonucleotide portion of themacromolecule this will impart nuclease stability to the macromolecule.Further this will also impart increase binding and specificity to acomplementary strand.

Again, while not wishing to be bound by any particular theory, I haverecognized certain criteria that must be met for RNase H to recognizeand elicit cleavage of a RNA strand. The first of these is that the RNAstand at the cleavage site must have its nucleosides connected via aphosphate linkage that bears a negative charge. Additionally, the sugarof the nucleosides at the cleavage site must be a β-pentofuranosyl sugarand also must be in a 2′ endo conformation. The only nucleosides(nucleotides) that fit this criteria are phosphodiester,phosphorothioate, phosphorodithioate, phosphoroselenate andphosphorodiselenate nucleotides of 2′-deoxy-erythro-pento-furanosylS-nucleosides.

In view of the above criteria, even certain nucleosides that have beenshown to reside in a 2′ endo conformation (e.g., cyclopentylnucleosides) will not elicit RNase H activity since they do notincorporate a pentofuranosyl sugar. Modeling has shown thatoligonucleotide 4′ -thionucleosides also will not elicit RNase Hactivity, even though such nucleosides reside in an envelopeconformation, since they do not reside in a 2′ endo conformation.Additionally, since α-nucleosides are of the opposite configuration fromβ-pentofuranosyl sugars they also will not elicit RNase H activity.

Nucleobases that are attached to phosphate linkages via non-sugartethering groups or via non-phosphate linkages also do not meet thecriteria of having a β-pentofuranosyl sugar in a 2′ endo conformation.Thus, they likely will not elicit RNase H activity.

For incorporation into the 2′-deoxyoligonucleotide portion of themacromolecule of the invention, nucleosides will be blocked in the 5′position with a dimethoxytrityl group, followed by phosphitylation inthe 3′ position as per the tritylation and phosphitylation proceduresreported in Oligonucleotides and Analogs, A Practical Approach,Eckstein, F., Ed.; The Practical Approach Series, IRL Press, New York,1991. Incorporation into oligonucleotides will be accomplished utilizinga DNA synthesizer such as an ABI 380 B model synthesizer usingappropriate chemistry for the formation of phosphodiester,phosphorothioate or phosphorodithioate phosphate linkages as per thesynthetic protocols illustrated in Eckstein op. cit.

The 2′-deoxynucleotide subunits and the peptide nucleic acid subunits ofthe macromolecules of the invention are joined by covalent bonds to fixthe subunits of the macromolecule in the desired nucleobase sequence. Acovalent interconnection of a desired length is formed between each ofthe two adjacent regions of the macromolecule. Preferably a covalentinterconnection is achieved by selecting a linking moiety that can forma covalent bond to both of the different types of subunit moietiesforming the adjacent regions. Preferably the linking moiety is selectedsuch that the resulting chain of atoms between the linking moiety andthe different types of moieties is of the same length. In one preferredembodiment of the invention, particularly useful as a linkage thatinterconnect the 2′-deoxynucleotide and peptide nucleic acid subunitsare amide linkages. In other embodiments amine and ester linkages can beused.

The peptide nucleic acid subunit portions (the PNA portions) of themacromolecules of the invention have the general formula (I):

wherein:

n is at least 2,

each of L¹-L^(n) is independently selected from the group consisting ofhydrogen, hydroxy, (C₁-C₄)alkanoyl, naturally occurring nucleobases,non-naturally occurring nucleobases, aromatic moieties, DNAintercalators, nucleobase-binding groups, heterocyclic moieties, andreporter ligands, at least one of L¹-L^(n) being a naturally occurringnucleobase, a non-naturally occurring nucleobase, a DNA intercalator, ora nucleobase-binding group;

each of R³ and R⁴ is independently selected from the group consisting ofhydrogen, (C₁-C₄)alkyl, hydroxy- or alkoxy- or alkylthio-substituted(C₁-C₄)alkyl, hydroxy, alkoxy, alkylthio and amino;

each of C¹-C^(n) (CR⁶R⁷)_(y) where R⁶ is hydrogen and R⁷ is selectedfrom the group consisting of the side chains of naturally occurringalpha amino acids, or R⁶ and R⁷ are independently selected from thegroup consisting of hydrogen, (C₂-C₆)alkyl, aryl, aralkyl, heteroaryl,hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, NR³R⁴ and SR⁵, where R³ and R⁴are as defined above, and R⁵ is hydrogen, (C₁-C₆)alkyl, hydroxy-,alkoxy-, or alkylthio-substituted (C₁-C₆)alkyl, or R⁶ and R⁷ takentogether complete an alicyclic or heterocyclic system;

each of D¹-D^(n) is (CR⁶R⁷)_(z) where R⁶ and R⁷ are as defined above;

each of y and z is zero or an integer from 1 to 10, the sum y+z beinggreater than 2 but not more than 10;

each of G¹-G^(n−1) is —NR³CO—, —NR³CS—, —NR³SO— or —NR³SO₂—, in eitherorientation, where R³ is as defined above;

each pair of A¹-A^(n) and B¹-B^(n) are selected such that:

(a) A is a group of formula (IIa), (IIb) or (IIc) and B is N or R³N⁺; or

(b) A is a group of formula (IId) and B is CH;

where:

X is O, S, Se, NR³, CH₂ or C(CH₃)₂;

Y is a single bond, O, S or NR⁴;

each of p and q is zero or an integer from 1 to 5, the sum p+q being notmore than 10;

each of r and s is zero or an integer from 1 to 5, the sum r+s being notmore than 10;

each R¹ and R² is independently selected from the group consisting ofhydrogen, (C₁-C₄)alkyl which may be hydroxy- or alkoxy- oralkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;

each of G¹-G^(n−1) is —NR³CO—, —NR³CS—, —NR³SO— or —NR³SO₂—, in eitherorientation, where R³ is as defined above;

Q is —CO₂H, —CONR′R″, —SO₃H or —SO₂NR′R″ or an activated derivative of—CO₂H or —SO₃H; and

I is —NHR″′R″″ or —NR″′C(O)R″″, where R′, R″, R″′ and R″″ areindependently selected from the group consisting of hydrogen, alkyl,amino protecting groups, reporter ligands, intercalators, chelators,peptides, proteins, carbohydrates, lipids, steroids, nucleosides,nucleotides, nucleotide diphosphates, nucleotide triphosphates,oligonucleotides, oligonucleosides and soluble and non-soluble polymers.

Preferred peptide nucleic acids have general formula (IIIa)-(IIIc);

wherein:

each L is independently selected from the group consisting of hydrogen,phenyl, heterocyclic moieties, naturally occurring nucleobases, andnon-naturally occurring nucleobases;

each R^(7′) is independently selected from the group consisting ofhydrogen and the side chains of naturally occurring alpha amino acids;

n is an integer from 1 to 60;

each of k, l, and m is independently zero or an integer from 1 to 5;

p is zero or 1;

R^(h) is OH, NH₂ or —NHLysNH₂; and

R^(i) is H or COCH₃.

Particularly preferred are compounds having formula (IIIa)-(IIIc)wherein each L is independently selected from the group consisting ofthe nucleobases thymine (T), adenine (A), cytosine (C), guanine (G) anduracil (U), k and m are zero or 1, and n is an integer from 1 to 30, inparticular from 4 to 20.

The peptide nucleic acid portions of the macromolecules of the inventionare synthesized by procedures, either in solution or on a solid phase,generally following the procedures described in patent applicationPCT/EP/01219 that published on Nov. 26, 1992 as publication WO 92/20702or U.S. patent application 08/088,658, filed Jul. 2, 1993 or byequivalent procedures. The contents of these patent applications areherein incorporated by reference.

The synthons used are monomer amino acids or their activatedderivatives, protected by standard protecting groups. The PNAs also canbe synthesized by using the corresponding diacids and diamines.

The novel monomer synthons according to the invention are selected fromthe group consisting of amino acids, diacids and diamines having generalformulae:

wherein L, A, B, C and D are as defined above, except that any aminogroups therein may be protected by amino protecting groups; E is COOH,CSOH, SOOH, SO₂OH or an activated derivative thereof; and F is NHR³ orNPgR³, where R³ is as defined above and Pg is an amino protecting group.

Preferred monomer synthons according to the invention have formula(VIIIa)-(VIIIc):

or amino-protected and/or acid terminal activated derivatives thereof,wherein L is selected from the group consisting of hydrogen, phenyl,heterocyclic moieties, naturally occurring nucleobases, andnon-naturally occurring nucleobases; and R^(7′) is selected from thegroup consisting of hydrogen and the side chains of naturally occurringalpha amino acids.

Compounds of the invention can be utilized in diagnostics, therapeuticsand as research reagents and kits. Further once identified as beingactive in a test system, they can be used as standards in testingsystems for other active compounds including chemotherapeutic agents.They can be utilized in pharmaceutical compositions by including aneffective amount of a macromolecule of the invention admixed with asuitable pharmaceutically acceptable diluent or carrier. They furthercan be used for treating organisms having a disease characterized by theundesired production of a protein. The organism can be contacted with amacromolecule of the invention having a sequence that is capable ofspecifically hybridizing with a strand of nucleic acid that codes forthe undesirable protein.

Such therapeutic treatment can be practiced in a variety of organismsranging from unicellular prokaryotic and eukaryotic organisms tomulticellular eukaryotic organisms. Any organism that utilizes DNA-RNAtranscription or RNA-protein translation as a fundamental part of itshereditary, metabolic or cellular control is susceptible to suchtherapeutic and/or prophylactic treatment. Seemingly diverse organismssuch as bacteria, yeast, protozoa, algae, all plant and all higheranimal forms, including warm-blooded animals, can be treated by thistherapy. Further, since each of the cells of multi-cellular eukaryotesalso includes both DNA-RNA transcription and RNA-protein translation asan integral part of their cellular activity, such therapeutics and/ordiagnostics can also be practiced on such cellular populations.Furthermore, many of the organelles, e.g., mitochondria andchloroplasts, of eukaryotic cells also include transcription andtranslation mechanisms. As such, single cells, cellular populations ororganelles also can be included within the definition of organisms thatare capable of being treated with the therapeutic or diagnosticoligonucleotides of the invention. As used herein, therapeutics is meantto include both the eradication of a disease state, killing of anorganism, e.g., bacterial, protozoan or other infection, or control oferratic or harmful cellular growth or expression.

For purpose of illustration, the compounds of the invention are used ina ras-luciferase fusion system using ras-luciferase transactivation. Asdescribed in U.S. patent application Ser. No. 07/715,196, filed Jun. 14,1991, abandoned, entitled Antisense Inhibition of RAS Oncogene andassigned commonly with this application, the entire contents of whichare herein incorporated by reference, the ras oncogenes are members of agene family that encode related proteins that are localized to the innerface of the plasma membrane. Ras proteins have been shown to be highlyconserved at the amino acid level, to bind GTP with high affinity andspecificity, and to possess GTPase activity. Although the cellularfunction of ras gene products is unknown, their biochemical properties,along with their significant sequence homology with a class ofsignal-transducing proteins known as GTP binding proteins, or Gproteins, suggest that ras gene products play a fundamental role inbasic cellular regulatory functions relating to the transduction ofextracellular signals across plasma membranes.

Three ras genes, designated H-ras, K-ras, and N-ras, have beenidentified in the mammalian genome. Mammalian ras genes acquiretransformation-inducing properties by single point mutations withintheir coding sequences. Mutations in naturally occurring ras oncogeneshave been localized to codons 12, 13, and 61. The sequences of H-ras;K-ras and N-ras are known. Capon et al., Nature 302 1983, 33-37; Kahn etal., Anticancer Res. 1987, 7, 639-652; Hall and Brown, Nucleic AcidsRes. 1985, 13, 5255-5268. The most commonly detected activating rasmutation found in human tumors is in codon 12 of the H-ras gene in whicha base change from GGC to GTC results in a glycine-to-valinesubstitution in the GTPase regulatory domain of the ras protein product.Tabin, C. J. et al., Nature 1982, 300, 143-149; Reddy, P. E. et al.,Nature 1982, 300, 149-152; Taparowsky, E. et al., Nature 1982, 300,762-765. This single amino acid change is thought to abolish normalcontrol of ras protein function, thereby converting a normally regulatedcell protein to one that is continuously active. It is believed thatsuch deregulation of normal ras protein function is responsible for thetransformation from normal to malignant growth. Monia, et. al., J. Bio.Chem., 1993, 268, 14514-14522, have recently shown, via atransactivation reporter gene system, that chimeric “Gap” (structurehaving a 2′-deoxyoligonucleotide flanked by non-deoxyoligonucleotides)are active in vitro against the Ha-ras oncogene. Compounds of theinvention active in the above described assays can be used as standardsin in vitro chemotherapeutic agent test screens.

Compounds of the invention can be prepared via both solid phasesynthesis or solution phase synthesis. Both methods are illustrated inthe examples. Shown in the examples, in Example 1 is a general syntheticpreparation of the 2′-deoxyoligonucleotide portion of the macromoleculesof the invention. The schemes of Examples 2, 3 and 4 are illustrated inFIG. 1. Example 2 illustrates describes loading of the carboxy terminusof the right side PNA portion of the macromolecule to a solid supportresin. Example 3 describes elongation of this right side PNA portion ofthe macromolecule and formation of an amide linkage to a first2′-deoxynucleotide via a 3′-carboxy nucleoside. Example 4 illustratesthe elongation of the central 2′-deoxyoligonucleotide portion of themacromolecule including addition of 5′-aminonucleotide to effect anamide linkage to the second PNA (left side) portion of themacromolecule. The schemes of Examples 5 and 6 are shown in FIG. 2.Example 5 shows completion of the left side PNA portion. Example 6illustrated removal of the blocking groups and removal from the resin.The scheme of Example 7 is shown in FIG. 3. Example 7 illustrates theformation of a solution phase DNA linkages for positioning of a5′-amino-3′-nucleotide as the 5′-terminal nucleotide. The schemes ofExamples 8 and 9 are shown in FIGS. 4 and 5, respective. Example 8illustrates solution phase the solution phase coupling of a PNA portionof a macromolecule of the invention to the DNA portion. In this example,the oligonucleotide of Example 7 is coupled to a first “T” PNA subunit;whereas, in Example 9 a first “A” PNA subunit is coupled to the2′-deoxyoligonucleotide portion of the macromolecule. The schemes ofExamples 10, 11 and 12 are shown in FIG. 6. Example 10, 11 and 12 areillustrative of the solution phase coupling of a DNA portion of amacromolecule of the invention to a PNA portion.

In the below examples the subunits, irrespective of whether they arepeptide nucleic acid (PNA) groups or 2′-deoxy-nucleotides (DNA) groups,the subunits are identified using the standard capital one letterdesignations, i.e. A, G, C or T. Such designation is indicative of thenucleobase incorporated in to the subunits. Thus “A” is used both toidentify a 2′-deoxyadenosine nucleotide as well as a peptide nucleicacid subunit that include an adenine base. For the peptide nucleic acidsubunit the adenine base is attached to the N-(2-aminoethyl)glycinebackbone via carboxymethyl linker. A standard nucleotide linkage, i.e.,phosphodiester, phosphorothioate, phosphorodithioate orphosphoroselenate, is indicated by a hyphen ( - ) between two adjacentidentification letters, e.g. A-T indicates an adenosine nucleotidelinked to a thymidine nucleotide. To indicate a peptide nucleic acidlinkage either a “(p)” or a “(pna)” are utilized, e.g. A(p)-T indicatesan adenine peptide nucleic acid unit attached to a thymine peptidenucleic acid unit.

Transition linkages between 2′-deoxynucleotides and peptide nucleic acidunits are indicated in brackets. Thus “T-(3′-carboxy)-A” indicated athymidine nucleoside having a carboxy group at its 3′ position that islinked to the amine moiety of the 2-aminoethyl portion of adeninepeptide nucleic acid subunit. Whereas “A-(5′-amino)-T” would indicate athymidine nucleotide having an amine at its 5′ position that is linkedto the carboxyl moiety of the glycine portion of the adjacent thyminepeptide nucleic acid subunit. Terminal groups, e.g. carboxy, hydroxyl,N-acetylglycine and the like, are indicated using appropriate symbolicnomenclature.

Carbobenzoxy blocking groups are shown as a superscript Z, e.g., ^(Z).Phenoxyacetyl protecting group are shown as a superscript PAC, e.g.,^(PAC). As alternatives for the standard polystyrene Merrifield resindescribed, a highly cross-linked polystyrene sold by Pharmacia or apolyethyleneglycol/polystyrene graft copolymer called Tentagel sold byRapp Polymere might be used. To conveniently remove the macromoleculesof the invention, the glycine should be attached to the resin via anester linkage.

The following examples and procedures illustrate the present inventionand are not intended to limit the same.

EXAMPLE 1

Oligonucleotide synthesis:

Oligonucleotide portions of the macromolecules of the invention aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidate chemistry with oxidation by iodine.For phosphorothioate oligonucleotides, the standard oxidation bottle isreplaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide inacetonitrile for the step wise thiation of the phosphite linkages. Thethiation wait step was increased to 68 sec and was followed by thecapping step. Unless otherwise indicated, after cleavage from the CPGcolumn and deblocking in concentrated ammonium hydroxide at 55° C. (18hr), the oligonucleotides are purified by precipitation twice out of 0.5M NaCl solution with 2.5 volumes ethanol. Analytical gel electrophoresisis effected in 20% acrylamide, 8 M urea, 454 mM Tris-borate buffer,pH=7.0. Phosphodiester and phosphorothioate oligonucleotides are judgedfrom polyacrylamide gel electrophoresis as to material length.

EXAMPLE 2

Low Load t-butyloxycarbonylglycyl Merrifield resin

Hydroxymethyl polystyrene resin (1 g, 650 micromoles hydroxyl/g) wasplaced in a solid-phase peptide synthesis vessel and washed sequentially(1 minute shaking for each wash) with dichloromethane (DCM, 2 times 10mL), N,N-dimethylformamide (DMF, 2 times 10 mL), and acetonitrile (2times 40 mL). To a round-bottom flask was addedN-t-butyloxycarbonylglycine (701 mg, 4 mmoles) andO-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(1.156 g, 3.6 mmoles). Anhydrous acetonitrile (40 mL) was added to thevial followed by N,N-diisopropylethylamine (1.392 mL, 8 mmoles). Theflask was shaken until all solids were dissolved. After one minute thecontents of the vial were added to the peptide synthesis vessel andshaken for 125 minutes. The reaction solution was then drained away andthe support washed with acetonitrile (1 times 40 mL), pyridine (2 times40 mL) and DMF (2 times 40 mL). A solution of 10% (v/v) acetic anhydridein DMF (40 mL total volume) was added to the resin and the reactionshaken for 40 minutes. After draining off the reaction solution, theacetic anhydride cap was repeated as above. At the end of the secondcapping reaction the resin was washed with DMF (2 times 40 mL), pyridine(1 times 40 mL), and DCM (3 times 40 mL). The resin was then dried byblowing with argon.

The extent of glycine derivatization was determined by a quantitativeninhydrin assay. An aliquot of the above resin (50 mg) was placed in asolid-phase peptide synthesis vessel and washed with DCM (2 times 3 mL).The resin was then treated three times with a solution of 5% (v/v)m-cresol in trifluoroacetic acid (3 mL) with shaking for two minuteseach time. After draining off the third reaction solution, the resin waswashed with DCM (3 times 3 mL), pyridine (3 times 3 mL), and DCM (3times 3 mL). The resin was then dried by blowing with argon.

An aliquot (5 mg) of the deprotected dried resin was placed in a testtube. To the resin were added a solution of 70% (v/v) water/pyridine (80microL), Kaiser reagent 1 (100 microL, 200 micromolar KCN in 2%H₂O/pyridine), reagent 2 (40 microL, 5% [w/v] ninhydrin in n-BuOH), andreagent 3 (50 microliters, 80% [w/v] phenol in n-BuOH). The reaction washeated at 100° C. for 10 minutes, cooled and diluted with 60% (v/v) EtOH(7 mL). The absorbance at 570 nm was then compared to a control reactioncontaining no resin and to a standard curve based on quantitation ofglycine ethyl ester. A derivatization level of 100 micromoles glycineper gram resin was obtained.

EXAMPLE 3

5′-Hydroxy-T(3′-carboxy)-T(p)-C^(z)(p)-A^(z)(p)-G^(z)(p)-Gly-O-Resin

t-Butyloxycarbonylglycyl Merrifield resin (200 mg, 10 microequivalents,example 1) is placed in a solid-phase peptide synthesis vessel. Thesupport is washed with 50% DMF/DCM (4 times 5 mL) and then treated twicewith 5% m-cresol in trifluoroacetic acid (4 mL) with shaking for twominutes each time. The support is washed again with 50% DMF/DCM (4 times5 mL) and then with pyridine (5 times 5 mL). To a vial are addedN²-benzyloxycarbonyl-1- (t-butyloxycarbonyl-aminoethylglycyl) guanine(80 micromoles) and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (72 micromoles). N,N-Dimethylformamide (0.4 mL) andpyridine (0.4 mL) are added to the vial followed byN,N-diisopropylethylamine (160 micromoles). The vial is shaken until allsolids are dissolved. After one minute the contents of the vial areadded to the peptide synthesis vessel and shaken for 20 minutes. Thereaction solution is then drained away and the support washed withpyridine (4 times 5 mL). Remaining free amine is capped by addition of a10% solution of N-benzyloxycarbonyl-N′-methylimidazole triflate inN,N-dimethylformamide (0.8 mL). After shaking for five minutes, thecapping solution is drained and the support washed again with pyridine(4 times 5 mL).

The deprotection, coupling, and capping as described in the aboveparagraph are repeated with three additional PNA monomers:N⁶-benzyloxycarbonyl-1-(t-butyloxycarbonyl-aminoethylglycyl)adenine,N⁴-benzyloxycarbonyl-1-(t-butyloxycarbonyl-aminoethylglycyl)cytosine,and 1-(t-butyloxycarbonyl-aminoethylglycyl)thymine.

The deprotection, coupling, and capping are then repeated once more, butthe monomer used in this case is5′-(tertbutyldiphenylsilyl)-3′-carboxymethylthymidine. After the cappingreaction, the resin is washed with pyridine (3 times 3 mL) and DMF (3times 3 mL). Anhydrous THF (3 mL) is added to the flask followed by 45microL of 70% (v/V) HF/pyridine. After shaking overnight the resin iswashed with THF (5 times 3 mL), DMF (3 times, 3 mL), acetonitrile (5times 3 mL), and DCM (5 times 3 mL). The resin is then dried by blowingwith argon.

EXAMPLE 4

T(5′-amino)-G^(PAC)-C^(PAC)-A^(PAC)-T-T(3′-carboxy)-T(p)-C^(z)(p)-A^(z)(p)-G^(z)(p)-Gly-O-Resin

5′-Hydroxy-T(3′-carboxy)-G^(z)(p)-C^(z)(p)-A^(z)(p)-T(p)-Gly-O-Resin (10microequivalents, example 3) is placed in a DNA synthesis column.Standard DNA synthesis (Example 1) is performed with phosphoramiditescontaining phenoxyacetyl protecting groups on the exocyclic amines,2-cyanoethyl groups on the phosphorous, and 4,4′-dimethoxytrityl groupson the 5′-hydroxyl. The 5′-terminal phosphoramidite coupled is5′-(monomethoxytritylamino)thymidine-3′-(N,N-diisopropylamino-2-cyanoethyl)phosphoramidite.The monomethoxytrityl group is removed by the standard automatedtreatment with dichloroacetic acid in DCM. After washing with DCM, theresin is dried under reduced pressure.

EXAMPLE 5

N-Acetylglycyl-T(p)-T(p)-C^(z)(p)-T(p)-C^(z)(p)-G^(z)(p)-C^(z)(p)-COOH

Hydroxymethyl polystyrene resin (115 mg, 75 microequivalents) was placedin a solid-phase peptide synthesis vessel. The support was washed withDCM (3 times 3 mL), DMF (3 times 3 mL), pyridine (3 times 3 mL), and DMFagain (2 times 3 mL). To a vial was added N⁴-benzyloxycarbonyl-1-(t-butyloxycarbonyl-aminoethylglycyl)cytosine (151 mg, 300 micromoles)and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(87 mg, 270 micromoles). N,N-Dimethylformamide (1.25 mL) and pyridine(1.25 mL) were added to the vial followed by N,N-diisopropylethylamine(105 microL, 600 micromoles). The vial was shaken until all solids weredissolved. After one minute the contents of the vial were added to thepeptide synthesis vessel and shaken for 30 minutes. The reactionsolution was then drained away and the support washed with DMF (4 times3 mL) The coupling of the C monomer was repeated as above. The resin wasthen capped by addition of 10% N-benzyloxycarbonyl-N′-methyl-imidazoletriflate in N,N-dimethylformamide (2.25 mL) followed by shaking for 5minutes. The resin was finally washed with pyridine (4 times 3 mL) andwas then ready for chain extension.

The support was washed with 50% DMF/DCM (4 times 3 mL) and then treatedtwice with 5% m-cresol in trifluoroacetic acid (3 mL) with shaking fortwo minutes each time. The support was washed again with 50% DMF/DCM (4times 3 mL) and then with pyridine (5 times 3 mL). To a vial were addedN²-benzyloxycarbonyl-1-(t-butyloxycarbonyl-aminoethylglycyl)guanine (163mg, 300 micromoles) and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (87 mg, 270 micromoles). N,N-Dimethylformamide (1.25mL) and pyridine (1.25 mL) were added to the vial followed byN,N-diisopropylethylamine (105 microL, 600 micromoles). The vial wasshaken until all solids were dissolved. After one minute the contents ofthe vial were added to the peptide synthesis vessel and shaken for 20minutes. The reaction solution was then drained away and the supportwashed with pyridine (5 times 3 mL). Remaining free amine was capped byaddition of a 10% solution of N-benzyloxycarbonyl-N′-methylimidazoletriflate in N,N-dimethylformamide (2.25 mL). After shaking for fiveminutes, the capping solution was drained and the support washed againwith pyridine (4 times 3 mL).

The deprotection, coupling, and capping as described in the aboveparagraph were repeated in the following order with the PNA monomers:N⁴-benzyloxycarbonyl-1-(t-butyloxycarbonyl-aminoethylglycyl)cytosine,and 1-(t-butyloxycarbonyl-aminoethylglycyl)thymine,N⁴-benzyloxycarbonyl-1-(t-butyloxycarbonylaminoethylglycyl)cytosine,1-(t-butyloxycarbonyl-aminoethylglycyl)thymine,1-(t-butyloxycarbonylaminoethylglycyl)-thymine, and then withN-acetylglycine. After the last capping reaction, the resin was washedwith pyridine (5 times 3 mL) and DCM (4 times 3 mL). The resin was thendried by blowing with argon.

A portion (29 mg, 10 microequivalents) of the resin was placed in asolid phase peptide synthesis vessel and tetrahydrofuran (2.5 mL) wasadded, followed by a solution of aqueous saturated potassium bicarbonate(0.25 mL) and tetrabutylammonium hydrogen sulphate (34 mg, 100micromoles). The reaction was then shaken for 14 hours at roomtemperature. Water (1 mL) was added to the reaction, causingprecipitation of a white solid. The liquid was filtered off and saved.The resin and white solid were then washed with additional water (1 mL)which was added to the first wash. Concentration to dryness underreduced pressure resulted in a white solid mixed with a pink oil. Water(6 mL) was added and the pH was adjusted to 2 with solid KHSO₄. Theliquid and suspended yellow solid were then transferred to Eppendorftubes and the solid was spun down. The solvent was removed and theresidual yellow solid was redissolved in 30% acetonitrile in watercontaining 0.1% TFA. Reverse phase chromatography resulted in thedesired product (2.6 mg, 1 micromole): molecular mass=2498 (electrospraymass spectrometry).

EXAMPLE 6

N-Acetylglycyl-T(p)-T(p)-C(p)-T(p)-C(p)-G(p)-C(p)-T(5′-amino)-G-C-A-T-T(3′-carboxy)-T(p)-C(p)-A(p)-G(p)-Gly-COOH

T(5′-Amino)-G^(PAC)-C^(PAC)-A^(PAC)-T-T(3′-carboxy)-T(p)-C^(z)(p)-A^(z)(p)-G^(z)(p)-Gly-O-resin(5 microequivalents, Example 3) is placed in a solid-phase peptidesynthesis vessel. The resin is washed with DCM (3 times 3 mL), DMF (3times 3 mL), and pyridine (3 times 3 mL). To a vial are addedN-Acetylglycyl-T(p)-T(p)-C^(z)(p)-T(p)-C^(z)(p)-G^(z)(p)-C^(z)(p)-COOH(10 micromoles, prepared as in example 5) andO-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (9micromoles). N,N-Dimethylformamide (0.3 mL) and pyridine (0.3 mL) areadded to the vial followed by N,N-diisopropylethylamine (20 micromoles).The vial is shaken until all solids are dissolved. After one minute thecontents of the vial are added to the peptide synthesis vessel andshaken for 4 hours. The reaction solution is then drained away and thesupport washed five times with pyridine.

Tetrahydrofuran (2 mL) is added to the resin, followed by a solution ofaqueous saturated potassium bicarbonate (0.2 mL) and tetrabutylammoniumhydrogen sulphate (27 mg, 80 micromoles). The reaction is shaken for 14hours at room temperature. The solution is then drained and kept. Theresin is washed with 50% tetrahydrofuran/water (3 times 1 mL) and withwater (3 times 2 mL). The wash solutions are added to the reactionsolution and concentrated under reduced pressure to removeorganics—concentration is stopped at a final volume of 2 milliliters.Inorganics are removed by gel filtration. The crude material isdissolved in aqueous 15 mM acetic acid (10 mL) and alternately degassedunder vacuum and back-filled with nitrogen four times. Palladium onBaSO₄ (5%, 30 mg) is added and the solution is stirred at RT ° C. for 2hours. The catalyst is removed by filtration and the product is thenpurified by reverse phase HPLC.

EXAMPLE 7

5′-Amino-deoxythymidylyl-(3′-5′)-3′-O-tertbutyldiphenylsilyldeoxythymidine(H₂N-T-T)

3′-tertbutyldiphenylsilyldeoxythymidine (58 mg, 120 micromoles) and5′-(p-methoxytriphenylmethylamino)-3′-[O-(2-cyanoethyl)-N,N-diisopropylaminophosphoramidyl]deoxythymidine(71 mg, 100 micromoles) were put in separate 10 mL round-bottom flasksand each co-evaporated once with anhydrous pyridine (2 mL) and twicewith anhydrous acetonitrile (1.5 mL). The compounds were then eachdissolved in anhydrous acetonitrile (0.75 mL) and combined. The reactionwas initiated by the addition of a solution of 0.4 M 1H-tetrazole inanhydrous acetonitrile (1 mL, 400 micromoles tetrazole). After stirring50 minutes at room temperature the reaction was quenched by pouring intoan oxidizing solution (10 mL of 0.43% I₂ in 90.54% THF, 0.41% pyridine,and 9.05% water).

The oxidation reaction was stirred an additional 40 minutes and pouredinto a separtory funnel containing dichloromethane (50 mL) and water (20mL). Residual iodine was removed by washing the organic layer with asolution of 0.3% sodium metabisulfite in water (95 mL). The organiclayer was then concentrated to a yellow oil by rotary evaporation underreduced pressure. The yellow oil was co-evaporated with toluene (2×15mL) under reduced pressure to remove residual pyridine.

The crude material was then dissolved in dichloromethane (6 mL) and thetrityl group was removed by addition of a solution of 3% trichloroaceticacid in dichloromethane (3 mL). After stirring 15 minutes at roomtemperature the reaction was quenched by pouring into a separatoryfunnel containing cold (4 IC) saturated aqueous sodium bicarbonate (10mL). Additional dichloromethane (20 mL) was added and the organic layerwas separated. A residual emulsion in the aqueous layer was broken up byaddition of more dichloromethane (20 mL). The two organic layers werethen combined and concentrated to dryness under reduced pressure. Thedesired product was purified from the resulting crude tan solid bypreparative silica TLC run in 20% (v/v) ethanol/chloroform, 0.2%N,N-diisopropylethylamine (49 mg, 63 micromoles, 63%): R_(f) (20% (v/v)ethanol/chloroform, 0.2% N,N-diisopropylethylamine)=0.05; ¹H NMR (200MHz, MeOH-d4) d 7.65 (m, 4H), 7.4 (m, 8H), 6.45 (m, 1H), 5.95 (m, 1H),4.6 (m, 1H), 4.1 (m, 2H), 3.9 (m, 1H), 3.6 (m, 1H), 3.2 (m, 1H), 2.9 (m,H), 2.5-2.0 (m, 4H), 1.89 (s, 6H), 1.1 (s, 9H); ³¹P NMR (MeOH-d4) d0.85; ESMS m/e 782.

EXAMPLE 8

N,N-Diisopropylethylammonium salt of DMT-O-T(pna)-CONH-T-T

H₂N-TpT (25 mg, 30 micromoles) was dissolved in 1:1 DMF/pyridine (0.8mL). To a separate vial were added1-[O-4,4′-dimethoxytrityl-hydroxyethylglycyl)thymine (23.5 mg, 40micromoles) and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (11.6 mg, 36 micromoles). The vial's contents weredissolved in 1:1 DMF/pyridine (0.8 mL) and N,N-diisopropylethylamine (14microL, 80 micromoles). After 5 minutes the activated ester solution wasadded to the H₂N-TpT solution. The reaction was stirred at roomtemperature for 80 minutes and quenched by the addition of ethyl alcohol(0.5 mL) After an additional 150 minutes the reaction mixture wasconcentrated to dryness under reduced pressure. The resulting solid waspurified by preparative silica TLC run in 20% (v/v) ethanol/chloroform,0.2% N,N-diisopropylethylamine (27 mg, 20 micromoles, 67%): R_(f) (20%(v/v) ethanol/chloroform, 0.2% N,N-diisopropylethylamine)=0.18; ¹H NMR(200 MHz, DMSO-d6) d 11.35 (m, 3H), 8.95 (br s, 0.3H), 8.72 (br s,0.7H), 7.79 (m, 2H), 7.61-7.19 (m, 22H), 6.91 (m, 4H), 6.36 (m, 1H),6.03 (m, 1H), 4.78 (m, 2H), 4.59 (s, 1H), 4.41 (m, 3H), 4.20 (s, 1H),3.93 (m, 2H), 3.65 (s, 2H), 3.18 (br s, 3H), 2.97 (m, 2H), 3.74 (s, 6H),3.42 (m, 4H), 2.01 (br s, 4H), 1.77 (m, 7H), 1.62 (s, 2H), 1.03 (m,15H); ¹³C NMR (DMSO-d6): 167.9, 164.6, 163.8, 158.2, 150.5, 144.9,143.0, 136.1, 135.7, 132.9, 130.2, 129.8, 128.1, 127.8, 126.8, 123.4,118.6, 113.4, 110.7, 110.1, 107.9, 86.3, 84.0, 83.0, 74.8, 74.5, 61.3,56.2, 55.1, 47.5, 26.8, 18.7, 12.1: ³P NMR (DMSO-d6) d −0.2, −1.05; ESMSm/e 1351.

EXAMPLE 9

N,N-Diisopropylethylammonium salt of tBoc-A^(z)(pna)-CONH-T-T

H₂N-TpT (19 mg, 24 micromoles) was dissolved in 1:1 DMF/pyridine (0.65mL). To a separate vial were addedN⁶-benzyloxycarbonyl-1-(t-butyloxycarbonyl-aminoethylglycyl)-adenine(12.7 mg, 24 micromoles) andO-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (7.1mg, 22 micromoles). The vial's contents were dissolved in 1:1DMF/pyridine (0.65 mL) and N,N-diisopropylethylamine (8.4 microL, 48micromoles). After 2 minutes the activated ester solution was added tothe H₂N-TpT solution. The reaction was stirred at room temperature for105 minutes and quenched by the addition of ethyl alcohol (0.5 mL).After an additional 120 minutes the reaction mixture was concentrated todryness under reduced pressure. The resulting solid was purified bypreparative silica TLC run in 20% (v/v) ethanol/chloroform, 0.2%N,N-diisopropylethylamine (6 mg, 5 micromoles, 21%): R_(f) (20% (v/v)ethanol/chloroform, 0.2% N,N-diisopropylethylamine) =0.07; ¹H NMR (200MHz, DMSO-d6) d 11.37 (s, 1H), 11.22 (s, 1H), 10.64 (br s, 1H), 9.17 (brs, 0.3H), 8.90 (br s, 0.7H), 8.59 (m, 2H), 8.30 (m, 1H), 7.80 (M, 1H),7.58 (S, 3H), 7.38 (m, 12H), 7.16 (m, 1H), 6.36 (m, 1H), 6.04 (m, 1H),5.43 (m, 1H), 5.32 (s, 1H), 5.23 (s, 2H), 5.1 (s, 1H), 4.42 (m, 3H),4.19 (m, 1H), 3.98 (s, 1H), 3.87 (m, 3H), 3.77 (m, 1H), 3.66 (m, 1H),3.02 (m, 2H), 22.68 (m, 1H), 2.03 (m, 4H), 1.74 (m, 6H), 1.02 (m, 30H);³¹P NMR (DMSO-d6) d −0.01, -0.8.

EXAMPLE 10

1-(t-Butyloxycarbonyl-aminoethylglycyl)thymine, ethyl ester

1-(t-butyloxycarbonyl-aminoethylglycyl)thymine (300 mg, 780 micromoles)was dissolved in 50% DMF/pyridine (3.0 mL) and N,N-diisopropylethylamine(0.25 mL, 1.44 mmoles) was added. After stirring 5 minutesO-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (350mg, 1.1 mmoles) was added to give light orange solution. The reactionwas stirred 30 minutes after which absolute ethanol (0.75 mL, 4.26mmoles) was added. After stirring an additional 90 minutes the reactionmixture was concentrated to an oil under reduced pressure. The oil wasdissolved in ethyl acetate (30 mL) and cooled to 5° C. The pure productprecipitated as a white solid which was collected by filtration (289 mg,701 micromoles, 90%): ¹H NMR (200 MHz, CDCl₃) d 8.33 (br s, 1H), 7.02(s, 0.3H), 6.97 (s, 0.7H), 5.58 (m, 1H), 4.59 (s, 1.4H), 4.43 (s, 0.6H),4.1 (s, 2H), 3.54 (m, 2H), 3.33 (m, 2H), 1.92 (s, 3H), 1.47 (s, 9H).

EXAMPLE 11

TBDPS-T-CONH-T(pna)-OEt

The ethyl ester of 1-(t-Butyloxycarbonyl-aminoethylglycyl)thymine (30mg, 72 microMol) was dissolved in 1.0 mL of trifluoroacetic acid andstirred at RT for 30 minutes. This was concentrated in vacuo and thenco-evaporated with 5 mL of toluene twice. The5′-O-tertbutyldiphenylsilyl-3′-carboxymethyldeoxyribothymidine (25 mg,48 microMol) was dissolved in 0.400 mL of a 1:1 DMF/pyridine solution.To this solution was added TBTU (21 mg, 65 microMol) and N,N-diisopropylethyl amine (25 microL, 144 microMol). The reaction became a pale orangecolor and was stirred for 30 minutes. The amine from the TFAdeprotection step was dissolved in 0.6 mL of DMF/Pyridine, added to theactivated carboxythymidine solution and stirred at room temperature forone hour. TLC analysis (20% MeOH/DCM) indicated that all of theactivated acid was consumed. The solution was concentrated in vacuo toan oil. The oil was purified on a 2 mm preparative TLC Plate (20 mm×20mm) with 20% MeOH/DCM as the eluting solvent. The least polar fractioncontained the PNA-DNA chimera providing the desired product as a whitesolid (25 mg, 36 micromoles 50%); ¹H NMR (200 MHz, CDCl₃) 9.8 (br s,1H), 9.6 (br s, 1 H), 7.7 (m, 4 H), 7.3 (m, 9 H), 7.0 (dd, 1 H), 6.2 (m,1 H), 4.5-3.4 (12 H), 2.9 (m, 1 H), 2.5-2.1 (m, 4 H), 1.5 (s, 3 H), 1.3(d, 6 H), 1.1 (s, 9 H); ¹³C NMR (CDCl₃): d11.764, 12.083, 12.391,14.144, 26.796, 27.055, 29.708, 35.232, 37.177, 37.788, 38.932, 48.298,61.486, 64.854, 84.476, 85.018, 111.110, 127.711, 129.980, 135.407,135.654, 140.976, 150.835, 151.555, 167.416, 172.192; ESMS m/e 817.

EXAMPLE 12

T-CONH-T(pna)-OEt

The above dimer (30 mg, 0.058 mmol) was desilylated by dissolving in 1ml dry THF and cooled to 0° C. To this was added 20 mL of 70%HF/Pyridine and 10 mL of a 1 M solution of tetrabutyl ammonium fluoridewas added and the reaction mixture stirred overnight. TLC (10% MeOH/DCM)indicated the reaction was complete. The solution was quenched with 1 mlof saturated NaHCO₃ and stirred until the gas evolution ceased. Theaqueous layer was diluted with an additional 5 ml of water and extracted2× with 3 ml of ethyl acetate. The organic layers were combined anddiscarded. The aqueous layer was evaporated to dryness resulting in amixture of the deprotected dimer and NaHCO₃. The mixture was suspendedin methanol and purified on two 20×20 0.5 mm preparative TLC plateseluting with 30% ethanol in chloroform. After the plates finishedeluting, they were dried and the fluorescent band scraped and extracted.The extract was filtered and evaporated to yield 12 mg (56% yield) ofthe deprotected dimer that was contaminated with a small amount oftetrabutylammonium fluoride. ¹H(CD₄OD): 1.0-1.5 (mm, 10 H); 1.5 (m, 2H); 1.9 (s, 6 H); 2.1-2.8 (mm, 6 H); 3.2-4.0 (mm, 15 H); 4.1-4.4 (m, 4H); 4.7 (s, 1 H); 6.1 (m, 1 H); 7.3 (s, 1 H); 8.0 (s, 1 H).

EXAMPLE 13

(5′-DMT)-A^(z)-T-(3′-carboxy)-T(pna) (including cyanoethoxy protectedphosphodiester linkage)

The deprotected dimer of Example 12 (12 mg, 0.0207 mmol) wasco-evaporated twice with anhydrous pyridine and twice with anhydrousacetonitrile. The resulting white solid was dissolved in 3 ml of 1:1acetonitrile:DMF. To this solution was added 1 ml of a 0.1 M solution ofadenosine phosphoramidite and 1 ml of 0.4 M 1H-tetrazole solution. Thiswas stirred at ambient temperature for 1 hr, and then an additional 1 mlof amidite was added and stirring continued for an additional hour. Atthe end of that time 10 ml of oxidizing solution (0.43% I₂ in 90.54%THF, 0.41% pyridine, and 9.05% water) was added and the reaction stirredfor 1 hour. The reaction was quenched with 25 ml of a 1 M solution ofsodium bisulfite solution. This solution was extracted with chloroform2×20 ml and the combined extracts were washed with another 25 ml portionof bisulfite solution, resulting in a slightly yellow organic phase. Thechloroform solution was dried over magnesium sulfate and concentrated toa yellow oil. The mixture was purified using 20×20 cm preparative TLCplates (2, 0.5 mm coating) eluting with 20% acetone in dichloromethane.The diastereomeric mixture of trimer was isolated as an oil weighing 25mg for a 93% yield; ¹H (CDCl₃): 1.2 (m, 13 H); 2.5-3.2 (mm 5 H); 3.4 (m,4 H); 3.7 (s, 6H); 4.1 (m, 1 H); 4.4 (m, 1 H); 5.2 (m, 1 H); 6.5 (m,1H); 6.8 (m, 4 H); 7.3 (m, 10 H); 7.5 (m, 3 H); 8.0 (d, 2 H); 8.2 (d, 2H); 8.7 (s, 1 H); 9.1 (s, 1 H); ³¹P (CDCl₃): 8.247, 8.116.

EXAMPLE 14

Stability of PNA oligomers to NH₄OH deprotection conditions.

In the first case a PNA oligomer containing a free amino terminus(H-TAT-TCC-GTC-ATC-GCT-CCT-CA-Lys-NH₂) (all PNA) was dissolved in 70%concentrated NH₄OH. The reaction was incubated at 23° C. and aliquotswere examined by reverse phase HPLC at the time points indicated below.In the second experiment a PNA oligomer with a glycyl-capped aminoterminus (H-Gly-TGT-ACG-TCA-CAA-CTA-Lys-NH₂) (all PNA) was dissolved in90% concentrated NH₄OH and heated in a sealed flask at 55° C.

The NH₄OH stability of the PNA oligomer containing a free amino terminuswas insufficient to allow the removal of base protecting groups from thePNA/DNA chimera. Capping the amino terminus with a glycyl group greatlyincreased the stability of the PNA to aqueous base. The glycyl-cappedPNA demonstrated only minimal degradation at the 11 hour time point with15% decomposition after 23 hours. The glycyl capped PNA is completelystable to conditions used to remove the phenoxyacetyl protecting groupand relatively stable to those used for the standard DNA base protectinggroups (benzoyl and isobutyryl amides). The results are shown in Table1.

TABLE 1 Remaining Remaining hours uncapped, PNA, 23° C. capped, PNA, 55°C. 1 97 100 2 92 99 4 85 5 97 6 75 8 60 11 92 23 85

EXAMPLE 15

Macromolecule Having Peptide Nucleic Acids Regions Flanking A Central2′-Deoxy Phosphorothioate Oligonucleotide Region Joined via Amine andEster linkages

A first region of peptide nucleic acids is prepared as per Example 2above. The peptide nucleic acids is prepared from the C terminus towardsthe N terminus using monomers having protected amine groups. Followingcompletion of the first peptide region, the terminal amine blockinggroup is removed and the resulting amine reacted with a3′-C-(formyl)-2′,3′-dideoxy-5′-trityl nucleotide prepared as per theprocedure of Vasseur, et. al., J. Am. Chem. Soc. 1992, 114, 4006. Thecondensation of the amine with the aldehyde moiety of the C-formylnucleoside is effected as per the conditions of the Vasseur, ibid., toyield an intermediate imine linkage. The imine linkage is reduced underreductive alkylation conditions of Vasseur, ibid., withHCHO/NaBH₃CN/AcOH to yield the nucleoside connected to the peptidenucleic acid via an methyl alkylated amine linkage. An internal 2′-deoxyphosphorothioate nucleotide region is then continued from thisnucleoside as per the protocols of Example 1. Peptide synthesis for thesecond peptide region is commenced by reaction of the carboxyl end ofthe first peptide nucleic acid of this second region with the 5′ hydroxyof the last nucleotide of the DNA region following removal of thedimethoxytrityl blocking group on that nucleotide. Coupling is effectedvia EDC in pyridine to form an ester linkage between the peptide and thenucleoside. Peptide synthesis is then continued to complete the secondpeptide nucleic acid region.

EXAMPLE 16

Macromolecule Having Peptide Nucleic Acids Regions Flanking A Central2′-Deoxy Phosphoroselenate Oligonucleotide Region

The synthesis of Example 15 is repeated except for introduction of thephosphoroselenate linkages in the 2′-deoxynucleotide portion of themacromolecule, oxidization is effected with 3H-1,2-benzothiaseleno-3-olas per the procedure reported by Stawinski, et al., Tenth InternationalRoundtable: Nucleosides, Nucleotides and Their Biological Evaluation,Sep. 16-20, 1992, Abstracts of Papers, Abstract 80.

EXAMPLE 17

Macromolecule Having Peptide Nucleic Acids Regions Flanking A Central2′-Deoxy Phosphorodithioate Oligonucleotide Region

The synthesis of Example 15 is repeated except for introduction of thephosphorodithioate linkages in the 2′-deoxynucleotide portion of themacromolecule, oxidization is effected utilizing the procedures ofBeaton, et. al., Chapter 5, Synthesis of oligonucleotidephosphorodithioates, page 109, Oligonucleotides and Analogs, A PracticalApproach, Eckstein, F., Ed.; The Practical Approach Series, IRL Press,New York, 1991.

PROCEDURE 1

ras-Luciferase Reporter Gene Assembly

The ras-luciferase reporter genes were assembled using PCR technology.Oligonucleotide primers were synthesized for use as primers for PCRcloning of the 5′-regions of exon 1 of both the mutant (codon 12) andnon-mutant (wild-type) human H-ras genes. The plasmids pT24-C3,containing the c-H-ras1 activated oncogene (codon 12, GGC→GTC), andpbc-N1, containing the c-H-ras proto-oncogene, were obtained from theAmerican Type Culture Collection (Bethesda, Md.). The plasmid pT3/T7luc, containing the 1.9 kb firefly luciferase gene, was obtained fromClontech Laboratories (Palo Alto, Calif.). The oligonucleotide PCRprimers were used in standard PCR reactions using mutant and non-mutantH-ras genes as templates. These primers produce a DNA product of 145base pairs corresponding to sequences −53 to +65 (relative to thetranslational initiation site) of normal and mutant H-ras, flanked byNheI and HindIII restriction endonuclease sites. The PCR product was gelpurified, precipitated, washed and resuspended in water using standardprocedures.

PCR primers for the cloning of the P. pyralis (firefly) luciferase genewere designed such that the PCR product would code for the full-lengthluciferase protein with the exception of the amino-terminal methionineresidue, which would be replaced with two amino acids, an amino-terminallysine residue followed by a leucine residue. The oligonucleotide PCRprimers used for the cloning of the luciferase gene were used instandard PCR reactions using a commercially available plasmid(pT3/T7-Luc) (Clontech), containing the luciferase reporter gene, as atemplate. These primers yield a product of approximately 1.9 kbcorresponding to the luciferase gene, flanked by unique HindIII andBssHII restriction endonuclease sites. This fragment was gel purified,precipitated, washed and resuspended in water using standard procedures.

To complete the assembly of the ras-luciferase fusion reporter gene, theras and luciferase PCR products were digested with the appropriaterestriction endonucleases and cloned by three-part ligation into anexpression vector containing the steroid-inducible mouse mammary tumorvirus promotor MMTV using the restriction endonucleases NheI, HindIIIand BssHII. The resulting clone results in the insertion of H-ras 5′sequences (−53 to +65) fused in frame with the firefly luciferase gene.The resulting expression vector encodes a ras-luciferase fusion productwhich is expressed under control of the steroid-inducible MMTV promoter.These plasmid constructions contain sequences encoding amino acids 1-22of activated (RA2) or normal (RA4) H-ras proteins fused in frame withsequences coding for firefly luciferase. Translation initiation of theras-luciferase fusion mRNA is dependent upon the natural H-ras AUGcodon. Both mutant and normal H-ras luciferase fusion constructions wereconfirmed by DNA sequence analysis using standard procedures.

PROCEDURE 2

Transfection of Cells with Plasmid DNA

Transfections were performed as described by Greenberg, M. E., inCurrent Protocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.,John Wiley and Sons, NY,) with the following modifications. HeLa cellswere plated on 60 mm dishes at 5×10⁵ cells/dish. A total pf 10 μg or 12μg of DNA was added to each dish, of which 1 μg was a vector expressingthe rat glucocorticoid receptor under control of the constitutive Roussarcoma virus (RSV) promoter and the remainder was ras-luciferasereporter plasmid. Calcium phosphate-DNA coprecipitates were removedafter 16-20 hours by washing with Tris-buffered saline [50 Mm Tris-Cl(pH 7.5), 150 mM NaCl] containing 3 mM EGTA. Fresh medium supplementedwith 10% fetal bovine serum was then added to the cells. At this time,cells are pre-treated with the macromolecules of the invention prior toactivation of reporter gene expression by dexamethasone.

PROCEDURE 3

Treatment of Cells

Following plasmid transfection, cells are washed with phosphate bufferedsaline prewarmed to 37° C. and Opti-MEM containing 5 μg/mLN-[1-(2,3-dioleyloxy)propyl]-N,N,N,-trimethylammonium chloride (DOTMA)is added to each plate (1.0 ml per well). Test compounds are added from50 μM stocks to each plate and incubated for 4 hours at 37° C. Medium isremoved and replaced with DMEM containing 10% fetal bovine serum and theappropriate test compound at the indicated concentrations and cells areincubated for an additional 2 hours at 37° C. before reporter geneexpression is activated by treatment of cells with dexamethasone to afinal concentration of 0.2 μM. Cells are harvested and assayed forluciferase activity fifteen hours following dexamethasone stimulation.

PROCEDURE 4

Luciferase Assays

Luciferase is extracted from cells by lysis with the detergent TritonX-100 as described by Greenberg, M. E., in Current Protocols inMolecular Biology, (F. M. Ausubel, R. Brent, R. E. Kingston, D. D.Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.), John Wiley andSons, NY. A Dynatech ML1000 luminometer Is used to measure peakluminescence upon addition of luciferin (Sigma) to 625 μM. For eachextract, luciferase assays are performed multiple times, using differingamounts of extract to ensure that the data is gathered in the linearrange of the assay.

PROCEDURE 5

Melting Curves

Absorbance vs temperature curves are measured at 260 nm using a Gilford260 spectrophotometer interfaced to an IBM PC computer and a GilfordResponse II spectrophotometer. The buffer contained 100 mM Na⁺, 10 mMphosphate and 0.1 mM EDTA, pH 7. Test compound concentration is 4 μM foreach strand determined from the absorbance at 85° C. and extinctioncoefficients calculated according to Puglisi and Tinoco, Methods inEnzymol. 1989, 180, 304-325. T_(m) values, free energies of duplexformation and association constants are obtained from fits of data to atwo state model with linear sloping baselines. Petersheim, M. andTurner, D. H., Biochemistry 1983, 22, 256-263. Reported parameters areaverages of at least three experiments. For some test compounds, freeenergies of duplex formation are also obtained from plots of T_(m) ⁻¹ vslog₁₀ (concentration). Borer, P. N., Dengler, B., Tinoco, I., Jr., andUhlenbeck, O. C., J. Mol. Biol., 1974, 86, 843-853.

PROCEDURE 6

Gel Shift Assay

The structured ras target transcript, a 47-nucleotide hairpin containingthe mutated codon 12, is prepared and mapped as described in Lima etal., Biochemistry 1991, 31, 12055-12061. Hybridization reactions areprepared in 20 μl containing 100 mM sodium, 10 mM phosphate, 0.1 mMEDTA, 100 CPM of T7-generated RNA (approximately 10 pM), and testcompound ranging in concentration from 1 pM to 10 μM. Reactions areincubated 24 hours at 37° C. Following hybridization, loading buffer wasadded to the reactions and reaction products are resolved on 20% nativepolyacrylamide gels, prepared using 45 mM tris-borate and 1 mM MgCl₂(TBM). Electrophoresis is carried out at 10° C. and gels are quantitatedusing a Molecular Dynamics Phosphorimager.

PROCEDURE 7

RNase H Analysis

RNase H assays are performed using a chemically synthesized 25-baseoligoribonucleotide corresponding to bases +23 to +47 of activated(codon 12, G→U) H-ras mRNA. The 5′ end-labeled RNA is used at aconcentration of 20 nM and incubated with a 10-fold molar excess of testcompound in a reaction containing 20 mM tris-Cl, pH 7.5, 100 mM KCl, 10mM MgCl₂, 1 mM dithiothreitol, 10 μg tRNA and 4 U RNasin in a finalvolume of 10 μl. The reaction components are preannealed at 37° C. for15 minutes then allowed to cool slowly to room temperature. HeLa cellnuclear extracts are used as a source of mammalian RNase H. Reactionsare initiated by addition of 2 μg of nuclear extract (5 μl) andreactions are allowed to proceed for 10 minutes at 37° C. Reactions arestopped by phenol/chloroform extraction and RNA components areprecipitated with ethanol. Equal CPMs are loaded on a 20% polyacrylamidegel containing 7M urea and RNA cleavage products are resolved andvisualized by electrophoresis followed by autoradiography. Quantitationof cleavage products is performed using a Molecular DynamicsDensitometer.

PROCEDURE 8

ras Transactivation Reporter Gene System

The expression plasmid pSV2-oli, containing an activated (codon 12,GGC→GTC) H-ras cDNA insert under control of the constitutive SV40promoter, was a gift from Dr. Bruno Tocque (Rhone-Poulenc Sante, Vitry,France). This plasmid is used as a template to construct, by PCR, aH-ras expression plasmid under regulation of the steroid-inducible mousemammary tumor virus (MMTV) promoter. To obtain H-ras coding sequences,the 570 bp coding region of the H-ras gene is amplified by PCR. The PCRprimers are designed with unique restriction endonuclease sites in their5′-regions to facilitate cloning. The PCR product containing the codingregion of the H-ras codon 12 mutant oncogene is gel purified, digested,and gel purified once again prior to cloning. This construction iscompleted by cloning the insert into the expression plasmid pMAMneo(Clontech Laboratories, Calif.).

The ras-responsive reporter gene pRDO53 is used to detect rasexpression. Owen et al., Proc. Natl. Acad. Sci. U.S.A. 1990, 87,3866-3870.

PROCEDURE 9

Northern blot analysis of ras expression in vivo

The human urinary bladder cancer cell line T24 is obtained from theAmerican Type Culture Collection (Rockville Md.). Cells are grown inMcCoy's 5A medium with L-glutamine (Gibco BRL, Gaithersburg Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells are seeded on 100 mm plates. Whenthey reached 70% confluency, they are treated with test compound. Platesare washed with 10 ml prewarmed PBS and 5 ml of Opti-MEM reduced-serummedium containing 2.5 μl DOTMA. Test compound is then added to thedesired concentration. After 4 hours of treatment, the medium isreplaced with McCoy's medium. Cells are harvested 48 hours after testcompound treatment and RNA is isolated using a standard CsClpurification method. Kingston, R. E., in Current Protocols in MolecularBiology, (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A.Smith, J. G. Seidman and K. Strahl, eds.), John Wiley and Sons, NY.

The human epithelioid carcinoma cell line HeLa 229 is obtained from theAmerican Type Culture Collection (Bethesda, Md.). HeLa cells aremaintained as monolayers on 6-well plates in Dulbecco's Modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mlpenicillin. Treatment with test compound and isolation of RNA areessentially as described above for T24 cells.

Northern hybridization: 10 μg of each RNA is electrophoresed on a 1.2%agarose/formaldehyde gel and transferred overnight to GeneBind 45 nylonmembrane (Pharmacia LKB, Piscataway, N.J.) using standard methods.Kingston, R. E., in Current Protocols in Molecular Biology, (F. M.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G.Seidman and K. Strahl, eds.), John Wiley and Sons, NY. RNA isUV-crosslinked to the membrane. Double-stranded ³²P-labeled probes aresynthesized using the Prime a Gene labeling kit (Promega, Madison Wis.).The ras probe is a SalI-NheI fragment of a cDNA clone of the activated(mutant) H-ras mRNA having a GGC-to-GTC mutation at codon-12. Thecontrol probe is G3PDH. Blots are prehybridized for 15 minutes at 68° C.with the QuickHyb hybridization solution (Stratagene, La Jolla, Calif.).The heat-denatured radioactive probe (2.5×10⁶ counts/2 ml hybridizationsolution) mixed with 100 μl of 10 mg/ml salmon sperm DNA is added andthe membrane is hybridized for 1 hour at 68° C. The blots are washedtwice for 15 minutes at room temperature in 2×SSC/0.1% SDS and once for30 minutes at 60° C. with 0.1×SSC/0.1%SDS. Blots are autoradiographedand the intensity of signal is quantitated using an ImageQuantPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). Northern blotsare first hybridized with the ras probe, then stripped by boiling for 15minutes in 0.1×SSC/0.1×%SDS and rehybridized with the control G3PDHprobe to check for correct sample loading.

PROCEDURE 10

Inhibition of proliferation of cancer cells and use as controls forchemotherapeutic agent test

Cells are cultured and treated with test compound essentially asdescribed in Example 9. Cells are seeded on 60 mm plates and are treatedwith test compound in the presence of DOTMA when they reached 70%confluency. Time course experiment: On day 1, cells are treated with asingle dose of test compound at a final concentration of 100 nM. Thegrowth medium is changed once on day 3 and cells are counted every dayfor 5 days, using a counting chamber. Dose-response experiment: Variousconcentrations of test compound (10, 25, 50, 100 or 250 nM) are added tothe cells and cells are harvested and counted 3 days later. The activecompounds of the invention can then be used standards in this samescreen for screening of other chemotherapeutic agents.

4 1 10 DNA Artificial Sequence misc_feature Antisense Sequence 1tgcatttcag 10 2 17 DNA Artificial Sequence misc_feature AntisenseSequence 2 ttctcgctgc atttcag 17 3 20 DNA Artificial Sequencemisc_feature Antisense Sequence 3 tattccgtca tcgctcctca 20 4 15 DNAArtificial Sequence misc_feature Antisense Sequence 4 tgtacgtcac aacta15

What is claimed is:
 1. A macromolecule of the structure: PNA-DNA-PNAwherein: said DNA comprises at least one 2′-deoxynucleotide; and each ofsaid PNAs comprise at least one peptide nucleic acid subunit.
 2. Amethod of in vitro modification of RNA, comprising contacting a testsolution containing RNase H and said RNA with a macromolecule of claim1, thereby effecting modification of said RNA.
 3. A method of in vitromodification of RNA, comprising contacting a test solution containingRNase H and said RNA with a compound having the structure: PNA-DNA-PNAwherein: said DNA comprises at least three 2′-deoxynucleotides linkedtogether in a sequence; and each of said PNAs comprise at least twopeptide nucleic acid subunits, wherein each of said PNAs comprises acompound of the formula IIIa, IIIb or IIIc:

wherein: each L is independently selected from the group consisting ofthe nucleobases thymine (T), adenine (A), cytosine (C), guanine (G), anduracil (U); each R⁷ is independently selected from the group consistingof hydrogen and the side chains of naturally occurring alpha aminoacids; n is an integer from 2 to 30; l, is independently zero or aninteger from 1 to 5; k and m are independently zero or 1; p is zero or1; R^(h) is OH, NH₂ or —NHLysNH₂; and R^(i) is H or COCH₃ 24, therebyeffecting modification of said RNA.
 4. The method of claim 2 whereinsaid contacting effects modification of said RNA.
 5. The method of claim3 wherein said contacting effects modification of said RNA.